PROJECT 2: ABSTRACT Cure or control of HIV infection in the absence of antiretroviral therapy (ART) will likely require host immune responses that eliminate HIV-infected cells or prevent new rounds of viral replication. A key problem is the ability of HIV to target and destroy CD4 T-cells that coordinate adaptive immune responses. Sangamo BioSciences together with the Hoxie lab at UPenn have shown that coreceptors (CoR) CXCR4 and CCR5, when modified to contain a C34 peptide from the gp41 HR2 domain conjugated to their amino termini, become potent trans-dominant inhibitors of HIV-1 entry. Inhibition occurs at ratios of <1:10 C34-conjugated to unconjugated CoR, respectively, and occurs irrespective of viral tropism, for multple clades, and for viruses that are resistant to soluble T20. The breadth and potency of HIV inhibition was demonstrated in vitro on primary CD4 T-cells where cells expressing C34-CoR showed a clear survival advantage during HIV-1 infection. We hypothesize that stable expression of C34-conjugated CoR on peripheral CD4 T-cells in vivo will lead to HIV resistance, enabling immune responses to control virus in the absence of ART and to target viral reservoirs following cessation of ART. This project will advance these observations with three Specific Aims: 1) The mechanisms underlying this extremely potent trans-dominant inhibition by C34-CoR will be explored by determining the role of CoR trafficking and positioning of the C34-peptide; 2) Resistance to HIV-1 has also been conferred using a novel gene editing approach to insert the C34 peptide into the endogenous CXCR4 gene, and will be studied in primary CD4 cells in vitro and in humanized mice when this C34-edited CoR is expressed under physiologic control of its endogenous promoter; and 3) Anticipated viral escape from C34- CoR inhibition will be assessed in vitro and in vivo to identify mutations, determine the underlying mechanisms, and assess barriers and fitness costs that can inform clinical studies in which C34-CoRs are expressed on autologous CD4 T-cells and used in cell-based therapeutic studies. The preliminary studies for Project 2 have provided the rationale for translating the potency of C34-CoR inhibition of HIV-1 to a human trial (Project 1), and its three Specific Aims will provide a mechanistic understanding of this effect at the cellular, virologic, and molecular levels that will enable further advances to be made. In addition, this approach for protecting CD4 T- cells from HIV infection, coupled with approaches to direct effector T-cells to virally-infected targets (Project 3) and to sustain these responses in vivo (Project 4), position this Program Project for developing a new paradigm of HIV control and possibly eradication.